Antifibrotic therapy disrupts stromal barriers and modulates the immune landscape in pancreatic ductal adenocarcinoma

Cell lines.

All murine primary cell lines were generated from freshly excised KPC tumors (the KPC genetically engineered mouse model is described in detail in following section) in our laboratory and were confirmed to be mycoplasma-free. Primary carcinoma cells were confirmed to have the correct genotype and to have undergone Cre-mediated recombination resulting in activation of the Kras oncogene and point mutant p53. To isolate primary carcinoma cells and activated PSCs (i.e. CAFs), tumors were washed with a 10% penicillin streptomycin solution, minced into small pieces, submerged in 2 mg/ml collagenase and 100U/mL hyaluronidase at 37°C for 30 minutes with agitation, washed and resuspended five times in DMEM, then plated onto FBS-coated tissue culture plates. Pancreatic stellate cells were isolated from cell mixtures by differential trypsinization of mixed populations of cells to highly enrich PSCs, followed by resuspension and incubation with in a biotinylated mouse E-cadherin antibody solution (PlusCellect Mouse E-cadherin⁺ Cancer/Stem Cells Kit) in combination with anti-β4 integrin and anti-CD11b, followed by resuspension and incubation with Dynabeads Biotin Binder (ThermoFisher Scientific) to remove carcinoma cells and macrophages. Following the initial purification differential trypsinization and selection with only E-cadherin beads was used to maintain high PSC purity as needed. Cells were used between 3–15 passages. Human primary PSCs were generated using an outgrowth method (28). De-identified resectable human PDA was finely minced. Tissues were initially cultured in DMEM (Invitrogen) with 20% FCS (“CP-medium”), then second, in KSF medium (Invitrogen), and third, Dresden modified DME medium (“Dresden-medium”), consisting of CP medium and KSF medium at a ratio of 2:1. All media was supplemented with penicillin (100 U/mL) and gentamicin (2.5 mg/mL)(Invitrogen). Differential trypsinization was implemented to isolate PSCs from human tumors, no carcinoma cells were detected in the culture that were confirmed to be mycoplasma-free. Cells were used within 10 passages. All human samples were de-identified and obtained through the UMN BioNet program in accordance with the US Department of Health and Human Services (HHS) Common Rule and University of Minnesota IRB approval.

Genetically engineered mouse model.

All animal studies were approved by the Institutional Animal Care and Use Committee of the University of Minnesota. In vivo studies utilized the Kras^G12D/+;p53^R172H/+;Pdx-1-Cre (KPC) genetically engineered mouse model, which has proven to be highly reflective of the human cognate condition genetically, clinically, and histopathologically (29). KPC mice conditionally express endogenous, physiologic levels of the respective oncogene and tumor suppressor gene mutations in pancreatic progenitor cells, resulting in the stochastic development and spontaneous progression of preinvasive ductal lesions to invasive and metastatic carcinomas. A subset of studies were done using fluorescent reporter KPC mice (i.e. Kras^G12D/+;p53^R172H/+;Pdx-1-Cre;ROSA^Tdtomato/+ (KPCT) or Kras^G12D/+;p53^R172H/+;Pdx-1-Cre;ZsGreen^+/+ (KPCG) as we have previously described (30)).

KPC study endpoint protocol.

At the endpoint of the drug studies, doxorubicin was injected i.v. 15–20 minutes prior to euthanasia to visualize small molecule distribution in tumors. This was followed by a terminal surgery, in which animals were anaesthetized using isofluorane and an intracardiac (i.c.) injection of high molecular weight (2000 kDa) FITC- or TRITC-conjugated lysine fixable dextran was administered 5 minutes prior to euthanasia to label perfused blood vessels, followed by a terminal i.c. injection of 10% formalin. The organs were harvested and fixed in 10% neutral buffered formalin, followed by dehydration in 70% ethanol before paraffin-embedding and sectioning (4 μm) for subsequent histochemical or immunohistochemical staining. Separate sections of each sample were frozen in O.C.T. compound.

Ultrasound for enrollment and monitoring of disease progression.

Weekly ultrasound was performed on KPC mice using the Vevo® 2100 Imaging System for both enrollment and disease monitoring. When tumors reached a diameter of 4–9mm, mice were randomly enrolled in study groups. The Vevo® 2100 software was used to reconstruct 3-D tumor volumes for quantification of tumor volume growth over time.

Drug treatment plans and dosages.

Unless specifically noted, the mice described in this study were enrolled in 10-day intervention studies using halofuginone, an analog of the plant alkaloid, febrifugine. A small subset of mice were enrolled in 24-hour intervention studies to evaluate early immune dynamics following one dose of 0.75 mg/kg halofuginone hydrobromide (Sigma Aldrich) administered intraperitoneal (i.p.), followed by euthanasia 24 hours later (n=3). Mice enrolled in the control group received no treatment, but were continually monitored, then euthanized after 10 days (n=6). Mice in the 10-day HF group were treated 3x/week, receiving a total of 5 doses (n=5).

Measuring interstitial fluid pressure.

Tumor pressures were measured as previously described (3). Briefly, interstitial fluid pressure (IFP) measurements were performed using a Millar Mikro-Tip system (pressure catheter transducer SPR-1000, 0.33 mm diameter) connected to a PCU-2000 Pressure Control Unit and the ADInstruments PowerLab data acquisition system (Millar Instruments, Inc.) The system was calibrated to 0, 25, and 100 mm Hg prior to each measurement following the manufacturer’s recommendations.

Histological analysis.

All stained tissue samples were digitally scanned at high resolution and viewed using Aperio ImageScope. Using this software we identified PDA-positive area within H&E stained sections. Likewise, this approach was used to identify and quantify necrotic area as a percentage of total PDA area. To quantify IHC stained tissue sections, ten regions of interest were randomly selected within the PDA-positive area, defined by referencing previously identified PDA-positive areas in adjacent H&E stained sections. Macros were written in FIJI to analyze each histological stain, using batch image processing to isolate and quantify the relevant positive signal. To identify perfused blood vessels, 2000 kDa FITC- (ThermoFisher Scientific) or TRITC-conjugated (Life Technologies) lysine-fixable dextrans were injected intracardiac (i.c.) 5 minutes prior to death, followed by a terminal i.c. injection of 10% formalin. Perfused blood vessels were imaged using an inverted Olympus IX81ZDC spinning disk confocal microscope. Corresponding regions were identified in adjacent tissue slices stained for CD31 and used to calculate total number of vessels. Percent perfusion was calculated as the number of fluorescently labeled, perfused blood vessels as a percent of the total number of CD31⁺ vessels in that region. To estimate immune infiltrate in H&E sections, a quantitative image-processing algorithm was created in FIJI. A built-in color deconvolution plugin was used to separate the H&E color channels and isolate the nuclei. Immune cells were distinguished by thresholding for specific nuclear characteristics (immune cells were presumed to have very small, round, and dark nuclei). Nuclear size parameters were determined by iterating the pixel range until the maximum particle size was small enough to exclude the visibly larger nuclei of carcinoma cells, and the minimum was small enough to exclude random debris. Circularity was set to 0.75–1.00 to exclude the elongated nuclei of CAFs. An example of the algorithm’s input and output is included in Figure S4.

Immunohistochemistry staining.

Formalin-fixed paraffin-embedded (FFPE) tissue sections were stained for αSMA (1:25; Dako, M0851), CD31 (1:50; Dianova, DIA310), iNos (1:150 Abcam; ab15323) and HTI-601 to stain for hyaluronan (1:500). For CD31 antigen retrieval was performed using 1X Citrate Buffer pH 6 (Sigma) for 20 min, washing with 1XTBS/0.1%Tween-20 (TBST)(all washes were done in TBST), blocking with 5% goat serum for 1 hr, then incubation with primary antibody overnight at 4°C. This was followed by an endogenous peroxidase block with 3% H₂O₂ for 15 min, incubation with a polymer secondary antibody for 30 min (Rat Probe, BioCare Rat-on-Mouse anti-CD31 Polymer Kit), followed by Rat-HRP (BioCare) for 30 min, and then counterstaining with freshly filtered Mayer’s Hematoxylin (Fisher Scientific) for 5 min, followed by dehydration and clearing with increasing concentrations of ethanol and xylenes. For αSMA, antigen retrieval was performed using a 1X Dako Tris/EDTA pH 9 solution for 20 min. This was followed with an endogenous peroxidase block of 3% H₂O₂ for 10 min, blocking for avidin/biotin (Vector kit), blocking with TCT buffer (0.1% trypsin, 0.1% CaCl₂, 20 mMTris-Cl pH 7.8) for 10 min, then incubation with primary antibody for 1 hour at RT. Slides were then incubated in MACH2 Rabbit HRP, followed by incubation with DAB for 10 min, counterstaining with hematoxylin, and finally dehydration and clearing. For iNos, antigen retrieval was performed using 1X Citrate Buffer pH 6 (Sigma) for 30 min, washing with 1XTBS/0.1%Tween-20 (TBST)(all washes were done in TBST), blocking with 5% goat serum for 1 hr, then incubation with primary antibody overnight at 4°C. This was followed by an endogenous peroxidase block with 3% H₂O₂ for 15 min, incubation with anti-rabbit HRP polymer secondary antibody for 30 min (BioCare Polymer Kit), followed incubation with DAB, and then counterstaining with freshly filtered Mayer’s Hematoxylin. To stain for HTI-601 (Halozyme): after de-waxing and rehydration, endogenous peroxide block of 0.3% H₂O₂ for 2 min, serum block of 2% FFA-BSA-2% normal goat serum for 30 min at room temperature, blocking with avidin/biotin (15 min each), incubation with primary antibody for 30 min at room temperature. For detection, incubate in HRP-streptavidin (BD Bioscience) for 15 min at room temperature, followed by incubation with DAB for 10 min, counterstaining with hematoxylin, then dehydration and clearing. All washes were done in TBST.

Immunofluorescent staining.

OCT frozen tissue sections were used to stain for CD11b (1:25; Abcam, ab8878), Ly6G (1:25; Bio X Cell, BP0075–1), and anti-pan cytokeratin (1:200; Millipore Sigma, F3418). Slides were air dried for 15 min at RT, incubated in acetone for 15 min, then air dried for 15 min. Slides were rehydrated with 1X PBS for 10 min, blocked with 2% normal goat serum for 1 hr at RT, and incubated with primary antibody for 2 hours at RT. Following this, secondary antibody (1:1000; Life Technologies,1749750) and directly-conjugated antibodies (PanCK) were added for 1 hour at RT, followed by counterstaining with Bisbenzimide (1:10,000; Sigma-Aldrich) for 10 min at RT, and mounting with Prolong Gold (Life Technologies). Washes between steps were done using 1XPBS. Immunfluorescence samples were imaged on a Nikon Ti-U fluorescence microscope. A subsection of FFPE tissue sections were sent to Reveal Biosciences (San Diego, CA) for IF staining, which were dual-stained for CD3/CD8, CD3/CD4, and CD4/FoxP3. For CD3/CD8 and CD3/CD4 staining, CD3 (1:100; Abcam) primary antibody was detected with Donkey anti-Rabbit IgG (H+L) secondary antibody, Alexa Fluor 546 conjugate (Life Technologies); CD8 (1:200; eBiosciences) primary antibody was detected with Goat anti-Rat IgG (H+L secondary antibody, Alexa Fluor 647 conjugate (Life Technologies); CD4 (1:200; eBiosciences) primary antibody was detected with Goat anti-Rat IgG (H+L secondary antibody, Alexa Fluor 647 conjugate (Life Technologies). Heat-induced antigen retrieval was performed with Epitope Retrieval Buffer 2 for 20 minutes. Primary antibodies were incubated overnight at 4°C. Non-specific background was blocked with 3% normal donkey serum + 3% normal goat serum in PBS-T. For CD4/FoxP3 staining, FoxP3 (1:100; Cell Signaling) primary antibody was detected with Donkey anti-Rabbit IgG (H+L secondary antibody, Alexa Fluor 546 conjugate (Life Technologies). CD4 (1:50; eBiosciences) primary antibody was directly conjugated to eFluor 660. Heat- induced antigen retrieval was performed using Leica Bond Epitope Retrieval Buffer 1 (Citrate solution, pH 6) or Epitope Retrieval Buffer 2 for 20 minutes. Primary antibodies were incubated overnight at 4°C. Non-specific background was blocked with 3% normal donkey serum in PBS-T. Whole slide fluorescent images were generated using a Pannoramic SCAN (3D Histotech). Cell staining for p53 was performed using the CM5 rabbit polyclonal antibody against p53 (1:200 Leica Biosystems; NCL-L-p53-CM5p) followed by Alexa Fluor 488 conjugate secondary antibody and Bisbenzimide.

Imaging and analysis of collagen fiber networks and small molecule distribution.

Multiphoton excitation microscopy and second harmonic generation (SHG) imaging were implemented to visualize doxorubicin distribution and fibrillar collagen in formalin-fixed paraffin embedded (FFPE) tissue sections that were rehydrated with xylenes and serial dilutions of ethanol, then mounted with Prolong Gold (Life Technologies). Visualization of collagen and doxorubicin was done on a custom-built multiphoton laser scanning microscope (Prairie Technologies/Bruker, Middleton, WI) that was described previously (30,31) using a Mai Tai Ti:Sapphire laser (SpectraPhysics, Santa Clara, CA) at an excitation wavelength of 880 nm and 800 nm, respectively.

TCGA human patient Cohort data analysis.

Transcript analysis was performed on human datasets publically available through the TDGA database. Correlation analysis was performed using the GEPIA platform (32).

Statistical analysis.

Two-group data were analyzed using a t-test. Multigroup data were analyzed using ANOVA followed by the Tukey multiple comparison post test. Percent FOV data was analyzed using Fisher’s exact test.

Biophysical barriers prevent effective drug delivery in PDA

As PDA progresses from preinvasive PanINs to invasive disease, an increasingly robust stromal environment develops, effectively preventing therapeutic distribution (4,9,12,33). This stromal complexity, which critically contributes to the unique pathology of PDA, is faithfully reflected in the genetically engineered Kras^LSL-G12D/+;p53^LSL-R172H/+;Pdx-1-Cre (KPC) mouse model (4,29,34)(Fig. 1A-T). Importantly, KPC tumors also utilize the TGF-β pathway (29), which contributes to the fibrotic and immunosuppressive desmoplastic reaction in both murine and human PDA (35,36), and activated PSCs from KPC tumors express transcripts reminiscent of fibrosis, e.g. Tgfβ1, Col1a1, and Fn1 (16). In KPC mice, α-SMA⁺ myofibroblasts emerge around early stage PanIN lesions and persist throughout later stages of disease (Fig 1A-C), depositing a robust ECM network (Fig. 1D-L). This includes a dense collagen matrix (Fig. 1D-I), which was confirmed to be largely fibrillar collagen from second harmonic generation (SHG) imaging (Fig. G-I), and large quantities of hyaluronan (Fig. 1J-L), a pervasive glycosaminoglycan that imbibes fluid (7,8), contributing to grossly elevated interstitial fluid pressure (IFP) in PDA (Fig. 1T), consistent with previous findings (4). When IFP increases to these levels, convective transvascular transport (i.e. intravascular to interstitial pressure-driven flux) is significantly hindered, and therapeutics must now be transported without, or even against, a pressure gradient. Moreover, compounding the unique hypovascularity of PDA (3,4)(Fig. 1S), many blood vessels actually collapse under the pressure (4), leaving a notable paucity of functional vessels in PDA, and to some degree in preinvasive disease (Fig. 1M-O). Indeed, CD31 immunohistochemistry (IHC) reveals a stark difference in blood vessel patency phenotypes between healthy pancreata with abundant normal vessels with apparent lumens, and pancreas tumors with a predominantly compressed vessel phenotype (i.e. no visible lumen)(Fig. 1M-O). Previously, we and others have shown that these phenotypes correlate with either fully or non-perfused vasculature, respectively (3,4,9). The collective impact of these stromal barriers on drug delivery can be visualized by analyzing doxorubicin distribution in normal pancreata, preinvasive, and invasive disease (Fig. 1P-R). A clear reduction in doxorubicin distribution and nuclear uptake is evident in diseased pancreata, and is increasingly pronounced with disease progression (Fig. 1Q,R).

Analysis of transcript data from the TCGA human PDA patient cohort likewise demonstrates robust expression of stromal ECM transcripts (Fig. 1U), as expected, e.g. (37,38). Further analysis reveals a significant correlation between α-SMA expression and expression of fibrotic ECMs, including type-I collagen, fibronectin, and hyaluronan synthases (Fig. 1V; Supplementary Fig. S1), with the strongest correlation between COL1A1 and α-SMA (ACTA2)(Supplementary Fig. S1). We note that high expression of type-I collagen, present in the majority of PDA patients, results in poorer survival outcome compared to the patient population with low expression (Fig. 1W). In fact, the 2-year survival for patients with low collagen expression levels is relatively high and surprisingly stable, suggesting that COL1A1 may be a useful biomarker to predict outcome, and that reducing collagen levels in PDA may be beneficial.

To better understand signal transduction associated with fibrosis, we next evaluated relationships between TGF-β signaling elements, which are elevated in human PDA compared to normal pancreata (Fig. 1U), and stromal ECM. Analysis demonstrates significant relationships between TGF-β pathway (i.e. SMAD2, SMAD3, TGFB1, and TGFB2) and stromal ECM transcripts (COL1A1, COL1A2, and HAS1, 2, and 3), with the strongest correlations for collagen (Figs. 1X-Z; Supplementary Fig S1). Similar relationships were observed between transcripts for type-I collagen or hyaluronan synthases and TGF-β receptors transcripts TGFBR1 or TGFBR2. Thus, these combined data in human and murine pancreas cancers suggest that targeting activated PSCs and collagen through the TGF-β pathway is a viable strategy to combat stromal fibrosis in PDA.

Halofuginone is a potent antifibrotic agent in autochthonous PDA

The potent antifibrotic efficacy of halofuginone (HF) have been demonstrated in pancreatitis and when administered near the onset of subcutaneously grafted pancreatic tumors (24,25). However, HF has yet to be evaluated in autochthonous PDA, particularly during intervention trials. First, to study of HF in vitro, primary PSCs derived from both human and KPC autochthonous tumors (Supplementary Fig. S2A-H) were treated with 0, 50, and 100 nM HF for 48 hours, demonstrating a significant, dose-dependent inhibition of per-cell α-SMA expression (i.e. PSC activation) in both mouse and human cells (Fig. 2A-C). Not surprisingly, inhibition of α-SMA expression corresponds to reduced expression of fibrosis-related genes, such as type-I collagen (Col1a1) and hyaluronan synthase 2 (Has2)(Fig. 2D), and consistent with reports targeting PSCs with Losartan (9,10). Note, since Has2 is expressed by activated PSCs at ~17X and ~85X the level of Has1 and Has3, respectively Supplementary Fig. S2I), we focused our analysis on Has2. Furthermore, both human and murine PSCs exhibit decreased proliferation at higher HF doses (Fig. 2E).

Compellingly, the antifibrotic effects of HF seen in vitro were maintained in vivo in physiologically relevant tumors. KPC mice were monitored and enrolled in 10-day intervention studies once tumors reached a diameter of 4–9mm in largest dimension, as evaluated using high-resolution ultrasound (Fig. 2F-M). Consistent with the results observed in vitro, mice treated with HF exhibited a significant decrease in stromal α-SMA⁺ signal, indicating a decrease in density of activated PSCs (Fig. 2F,G,N) and a profound effect on the stromal ECM. HF-treated animals showed a significant decrease in hyaluronan (Fig. 2H,I,O) and total collagen content, as determined through a Masson’s trichrome stain (Fig. 2J,K,P), with SHG imaging confirming this effect in fibrillar collagen (Fig. 2L,M,Q), underscoring the utility of HF as a potent antifibrotic agent in PDA.

Antifibrotic therapy facilitates drug delivery in established PDA

Hypertension is a characteristic property of virtually all solid tumors, disrupting natural pressure gradients and dramatically reducing convective transport (7,8,39). As a result, diffusive transport through the viscous, charged, tortuous, and sterically-challenging interstitial environments becomes the primary mechanism driving therapeutic distribution (8,39). We note that collagen content has been identified as a main determinant of interstitial transport, with hyaluronan also limiting transport by absorbing residual fluid, resulting in increased interstitial viscosity and intratumoral pressure (7,39). Therefore, we evaluated the impact of HF-induced depletion of collagen and hyaluronan on molecular transport in PDA. We found that stromal re-engineering to normalize tumor microenvironments with antifibrotic therapy markedly disrupts transport barriers associated with PDA (Fig. 3). IFP levels are significantly reduced in HF-treated animals (Fig. 3A), substantially increasing transvascular flux of small molecule therapy. Furthermore, relieving tumor pressure allows previously collapsed, nonfunctional vessels to open, leading to a significant increase in the percent of functional, perfused, vessels (Fig. 3B-F) without an increase in vessel density consistent with findings following PEGPH20 therapy but distinct from increased vessel density following Hh treatment (3,4). As a result of disrupting biophysical barriers to transport in PDA, a significant improvement in small molecule distribution is observed (Fig. 3G-O, Supplementary Fig. S3A,B), where doxorubicin distribution (Fig. 3P) negatively correlates with fibrotic area (Fig. 3Q). Thus, these data suggest that antifibrotic therapy with HF effectively disrupts barriers to therapeutic delivery and distribution, which is increasingly needed to enhance delivery of peptide and antibody therapies (e.g. molecular and immune therapies), and thus, may represent an effective component of carcinoma-stromal-immune combination therapy strategies.

Halofuginone performs as a dual-action therapy by targeting pancreatic carcinoma cells

HF is capable of inhibiting PSC activation and collagen production in vitro, even at very low, non-terminal levels (Fig. 2). At higher concentrations, PSCs exhibit dose-dependent inhibition of proliferation (Fig. 2). Interestingly, we also observe decreased proliferation of primary pancreatic carcinoma cells in vitro following HF treatment (Fig. 4A). This indicates the possibility of a dual-action role for HF as a PDA therapy in which carcinoma cells are impacted in addition to stroma-targeting effects. A number of targeted therapies have been reported to enhance carcinoma cell sensitivity to chemotherapy (40–42), including targeting CTGF, a member of the TGF-β signaling family (40). This, in combination with our proliferation data, led us to question whether HF might increase susceptibility of carcinoma cells to gemcitabine (Gem), a standard-of-care chemotherapeutic used to treat pancreatic cancer. To test this hypothesis, cells were subjected to various combination doses of HF and Gem, selected based on dose-response curves we established for each agent (Fig. 4A,B). Equal numbers of cells were allowed to adhere for 24 hours before HF-supplemented media was added for 24 hours. HF-supplemented media was then replaced by Gem-supplemented media for an additional 24 hours. Finally, supplemented media was removed and replaced with normal media for 2 hours, at which point a MTS cell proliferation assay assessed cell viability (Fig. 4C,D). Interestingly, the relative decrease in proliferation with increasing gemcitabine concentration remains fairly static (~66%), regardless of HF concentration used to pre-treat the cells, revealing that pre-treatment of carcinoma cells with HF does not increase sensitivity to gemcitabine (Fig. 4D). It is, however, important to note that gemcitabine treatment following HF therapy can further reduce the carcinoma cell population, suggesting that while HF may be an effective monotherapy, greater cell death may be achieved with HF in concert with chemo and/or immune therapies.

Antifibrotic therapy promotes influx and distribution of macrophages and cytotoxic T cells in PDA

Although immunotherapies are showing great promise in a number of cancers, their impact on PDA has remained modest. This is due, in part, to low neoantigen load, strongly immunosuppressive microenvironments, and a dense stroma, which can physically limit both drug and cell infiltration (2,5,6,43–45). However, studies that targeted the stroma in concert with checkpoint inhibition have observed strong immune response in PDA (16,45), demonstrating that a robust anti-tumor immune response can take place in PDA under the right therapeutic conditions. Thus, stromal re-engineering to normalize tumor microenvironments may not only facilitate delivery of therapeutics (to combat disease and aid antigen presentation), but also to promote distribution of immune therapies (e.g. checkpoint inhibition, cytokine blockade, etc.) and anti-tumor immune cell populations. Yet, our understanding of immune dynamics and surveillance following stroma targeting is currently limited. Therefore, to determine if HF results in immunomodulatory effects, we examined tissue from mice 24hr and 10-day after initiation of HF intervention therapy. Examination of H&E tumor sections suggested that mice treated with HF for only 24 hours exhibit a striking increase in immune infiltrate, an effect that appears to subside in the 10-day group (Fig. 5A-C). To quantify histopathology observations of immune infiltrate in H&E sections, we created an image-processing algorithm to distinguish and count immune cells based upon nuclear characteristics. The algorithm selected for nuclei that were smaller than the comparably larger nuclei of carcinoma cells, but larger than random debris, with circularity set to 0.75–1.00 to filter out the elongated nuclei of fibroblasts (Supplementary Fig. S4). During development, the validity of this algorithm was confirmed in a subset of slides stained for CD45. This analysis confirmed a statistically significant increase in immune infiltrate 24 hours after HF treatment that appears to normalize, in terms of cell numbers, by 10 days (Fig. 5D), demonstrating that HF treatment can alter the immune landscape in PDA. Despite this compelling result, this metric did not tell us which immune cell subpopulations were being altered, differences in immune composition between groups, or whether the composition in the 10-day group was altered. We therefore performed a more focused examination of distinct immune cell populations that have been implicated as key regulators of the immune response in PDA.

Inspection of specific immune populations (Fig. 5E-J) through immunofluorescence (IF) and IHC staining of KPC tumors (Supplementary Fig. S5A-R) revealed a significant increase in CD11b⁺ myeloid cells (Fig. 5E) and CD8⁺ cytotoxic T cells (Fig. 5J,K). Because CD11b is a broad myeloid cell marker, expressed on both protumorigenic (e.g. myeloid derived suppressor cells (MDSCs), “M2” alternatively activated macrophages) and anti-tumorigenic (e.g. “M1” classically activated macrophages) cells, we sought to further define this population. Analysis of IF staining for Ly6G, a validated marker for a primary immunosuppressive population of CD11b⁺ MDSCs in PDA (5), shows no significant difference in the number of Ly6G⁺ cells between groups (Fig 5F). However, there is an insignificant trend suggesting a decrease in Ly6G⁺ cells in both HF treatment groups, indicating that the increase in CD11b⁺ cells cannot be attributed to increased infiltration of immunosuppressive MDSCs. In contrast, analysis of IHC staining for iNOS, a functional marker of classically activated inflammatory macrophages, shows significant increase in the presence and distribution of iNOS⁺ cells following HF treatment (Fig. 5G, Supplementary Fig. 5G-I). The iNOS data is consistent with the trends observed in the Cd11b⁺ data, suggesting that inflammatory macrophages are a chief contributor to the immune influx observed at 24 hours and remain significantly elevated at 10 days, demonstrating a shift in the immune composition during HF treatment. Furthermore, analysis of T cell populations showed no significant difference for T helper cells (CD3⁺CD4⁺) or T regulatory cells (CD4⁺Foxp3⁺) (Fig. 5H,I), but reveals a significant increase in CD8⁺ cytotoxic T cells (Fig. 5J). This is particularly compelling when considered in the context of overall intratumoral distribution. Over half of the fields of view (FOV) analyzed in untreated tumors show a complete absence of CD8⁺ cells (Fig 5J), which may be related to reports that the robust ECM in PDA may impede T cell infiltration or distribution (46,47). In stark contrast, the 24hr HF group was completely negative for CD8⁺ cells in only 23% of FOVs (Fig. 5J), indicating a robust increase in the distribution of CD8+ T cells. These data show that targeting the stroma has the capacity to increases numbers, distribution, and thus sampling, of CD8⁺ T cells, which are capable of killing multiple cancer cells in a region. Thus, although the mere presence of intratumoral T cells does not guarantee an anti-tumor response, the ability to increase CD8⁺ cell infiltration in PDA is an indicator of overcoming two key immunosuppressive pathways in PDA, i.e. physical exclusion by ECM barriers and chemical exclusion via cytokines (16,45,47). Therefore, these data suggest that stromal re-engineering approaches can generate an enhanced anti-tumor immune response, but may require additional immune modulating therapy, including checkpoint inhibition, to enhance and maintain an anti-tumor immune response.

Regions of decreased hyaluronan are associated with increased immune infiltrate

To better understand the stromal changes that influence immune infiltrate into PDA, we revisited our investigation of the ECM (Fig. 6A-I). SHG imaging ruled out fibrillar collagen content as an explanation, since there is no significant difference between the 24hr and control groups (Fig. 6G-J). However, interestingly, we observed a “relaxation” of the collagen matrix, made evident by increased fiber waviness (i.e. greater crimp under low stress conditions that likely produce the trend of modestly increasing area fraction; Fig. 6H,J)). This is consistent with decreased myofibroblast behavior and decreased hyaluronan swelling pressures that are constrained by collagen networks (4,8), suggesting that the more relaxed collagen matrix may pose less of a barrier to immune cell infiltration and that changes in the hyaluronan matrix may be occurring. Indeed, we discovered a profound relationship between overall and regional hyaluronan levels and immune infiltrate (using nuclear phenotype algorithm analysis and visual confirmation in adjacent H&E sections) (Fig. 6A-F). In control mice, analysis of hyaluronan levels and regions of immune infiltration show an inverse correlation, with greater infiltrate associated with lower hylauronan (Fig. 6K,L). Moreover, we observed, perhaps surprisingly, that rapid changes in hylauronan emerge, with more regions containing low levels of hyaluronan just 24hr after HF treatment (Fig. 6D-F and 6J), suggesting that some degradation of hyaluronan has occurred. Interestingly, in the 24h group the relationship between hyaluronan levels and immune infiltration holds, and therefore greater frequency of hyaluronan-low areas in 24hr-group tumors are associated with a greater immune cell numbers, showing that decreased hyaluronan facilitates greater immune infiltration into PDA. Indeed, comparison of immune cells in “low HA” and “high HA” conditions (bottom and top 50% of signal for each group, respectively) demonstrates that there are greater numbers of immune cells in HA-low regions compared to HA-high regions for both the control and HF-treated mice, and significant increases in immune cells due to HF treatment (Fig. 6M), consistent with the increase in HA-low regions following HF treatment (Fig. 6E,K). Thus, we conclude that HF treatment results in a rapid impact on HA composition and, thereby, collagen matrix architecture (i.e. matrix loosening likely from decreased hyaluronan-generated swelling pressure), promoting immune infiltration into PDA.

Halofuginone treatment leads to widespread necrosis and reduced tumor volume growth

While HF alters immune dynamics and is an effective inhibitor of fibrosis and proliferation, macroscopic changes in tumor morphology speak to a more global, clinically-relevant outcome. To assess such changes, we measured tumor volume growth via high-resolution ultrasound in KPC mice enrolled in the 10-day intervention group (Fig. 7A-D). HF-treated mice exhibit significantly lower tumor volume growth compared to control mice, with one tumor decreasing in volume (Fig. 7E). Notably, and consistent with our quantitative measures of tumor volume, widespread necrosis is present in tumors from mice treated with HF for 10 days (Fig. 7F-H). Indeed, over 50% of the treated mice show necrosis in >20% of the tumor area, with one extreme responder displaying necrosis in over 50% of the evaluable tumor (Fig. 7H), with no observable toxicity or necrosis to adjacent normal pancreas (Supp. Fig 6A,B) or significant differences in body weight (Supp. Fig 6C). Overall, these data suggest that HF monotherapy significantly impacts PDA, but may be particularly useful in concert with single agent or combinations of molecular and/or immune therapies. Thus, we conclude that HF treatment results in reduced PSC activation inducing alterations in ECM levels and architecture and decreased carcinoma cell and CAF viability; this facilitates infiltration of anti-tumor immune populations, and in particular macrophages that may influence necrosis and help orchestrate T cell responses, and culminates with large regions of necrosis and reduced tumor growth. Therefore, while a series of reductionist and cell-depletion studies are required to parse out the specific impact of each cell population, data presented here clearly demonstrate that HF has strong potential to both improve drug delivery and promote infiltration of anti-tumor immune populations, warranting further study as part of a combination molecular and immune therapy approach to combat metastatic PDA.